DE2726899C1 - - Google Patents

Description

The invention is defined by the claims.

It is known that a number of Actinomycetes, especially Actinoplanaceae, inhibitors for glydoside hydrolases, preferably carbohydrate-cleaving enzymes of the digestive tract, form (DE-OS 20 64 092).

Furthermore, it is known that nojirimycin, a bacteriostatic antibiotic from strains of the genus Streptomyces, inhibits certain microbial α- glucosidases (T. NIWA et al. Agr. Biol. Chem. 34, 966 [1970]).

The present invention relates to inhibitors for glycoside hydrolases, in particular glucosidase inhibitors, that take effect in the digestive tract.

These inhibitors are from certain strains of the genus Bacillus formed (see. Claims 1 to 5).

To find the strains to be used according to the invention the methods described below were used.

Strains of are isolated in a known manner from earth samples  Bacillaceae family, in particular those of the genus Bacillus. Vaccinations of these strains are used to inoculate culture flasks with nutrient solutions that support the growth of these strains enable. You can e.g. B. use a nutrient solution that Contains 5 g peptone and 3 g meat extract per liter, however are basically many other types of nutrient solutions, the appropriate sources of carbon and nitrogen and contain nutrient salts, can be used. The pH of the Nutrient solutions can vary within wide limits; prefers an initial pH of the nutrient solution will be between 6.0 and 8.0 chosen.

They come as a carbon supplier for the nutrient solution various organic substances in question. Carbohydrates, organic acids and alcohols are for example called.

Yeast extract, soy flour, peptones, Meat extract and many other organic substances Find use.

The concentrations of carbon and nitrogen sources and the nutrient salts, of which FeSO₄, CaCO₃ and MgSO₄ are exemplary may fluctuate within wide limits. On the separate addition of nutrient salts can in in some cases, as they are often in the complex nitrogen sources as additives are.

Because the formation of inhibitors is often highly dependent on the composition the nutrient media is dependent, it is recommended the tribes to optimize production performance in different To cultivate nutrient solutions. Appropriate suggestions can be found in the examples.  

From the nutrient solution one fills z. B. 100-200 ml in one 1 liter Erlenmeyer flask, sterilized in a known manner, inoculates with the strain to be examined and incubates the piston at 15-80 ° C, preferably at 24-40 ° C or 50-70 ° C for thermophilic bacilli, on shaking machines. Shows the culture growth, what generally after 1-10 days, usually after 1-5 days, becomes visible, so you take a sample of z. B. 5 ml and separates in this Sample the cells by filtration or centrifugation from. From the culture broth 1-100 µl in the following described tests used and the inhibitory capacity calculated per ml.

The cells are filled twice with 5 volumes each (based on the cell volume) acetone and then once with 5 volumes Extracted diethyl ether. The combined extracts are evaporated to dryness, taken up in water and lyophilized. The lyophilisates are in concentrations of 10-1000 µg / ml in the tests described below used.

Amylase test

An amylase inhibitor unit (1 AIE) is defined as the amount of inhibitor, the two amylase units to 50% inhibited. An amylase unit (AE) is the amount of Enzyme in a minute below those listed below Test conditions 1 µ equivalent of glucosidic bonds splits in strength. The µVal split bonds reducing sugar formed as µVal with dinitrosalicylic acid determined colorimetrically and with the help of a  Maltose calibration curve given as µVal maltose equivalents. 0.1 ml of amylase solution is used to carry out the test (20-22 AU / ml) with 10-1000 µg inhibitor or 1-100 µl the culture solution to be tested in 0.4 ml of 0.02 M sodium glycerophosphate buffer / 0.001 M CaCl₂ pH 6.9 and about Equilibrated in a water bath at 35 ° C for 10-20 minutes. Then it is preheated to 35 ° C for 5 minutes with 0.5 ml 1% starch solution incubated at 35 ° C and then with 1 ml dinitrosalicylic acid reagent (according to P. Bernfeld in Colowick-Kaplan, Meth. Enzymol., Volume 1, page 149). The batch is opened for 5 minutes for color development heated in the boiling water bath, then cooled and with 10 ml of distilled water are added. The extinction at 540 nm becomes a correspondingly set blank value measured without amylase. For evaluation, a previously recorded amylase calibration curve after addition of inhibitor effective amylase activity read and from it the percentage inhibition of the amylase used is calculated. The percentage inhibition is a function of the Quotient

*) based on dry matter
**) AE in an uninhibited batch of the same series

applied, the 50% inhibition point read from the curve and converted to AIE / mg inhibitor.  

Sucrose test

A saccharase inhibitor unit (SIE) is defined as the amount of inhibitor, the two saccharase units to 50% inhibited. A sucrose unit (SE) is the amount of Enzyme in a minute below those listed below Test conditions 1 µmol sucrose in glucose and fructose splits. The micromoles of glucose formed are formed using the glucose oxidase reaction quantified under conditions in which further sucrose cleavage occurs the saccharase no longer takes place. To carry out 0.05 ml of the test is set to 0.12 SE Saccharase solution¹) with 1-20 µg inhibitor or 1-20 µl of the solution to be tested and mixed with 0.1 M sodium maleate buffer pH 6.0 to 0.1 ml.

1) Solubilized saccharase from porcine small intestinal mucosa after B. Borgström, A. Dahlquist, Acta Chem. Scand. 12, (1958), page 1997. With 0.1 M sodium maleate buffer pH 6.0 diluted to the appropriate SE content.

It is equilibrated at 35 ° C for 10 minutes and then with 0.1 ml of a 0.05 m sucrose solution preheated to 35 ° C added in 0.1 M sodium maleate buffer pH 6.0. Incubate 20 minutes at 35 ° C and stops the saccharase reaction by adding 1 ml of glucose oxidase reagent¹) and incubate for a further 30 minutes at 35 ° C. Then 1 ml 50% H₂SO₄ added and at 545 nm against a corresponding Blank value measured. For evaluation, the percentage inhibition of sucrose used calculated and from the 50% inhibition point using a glucose calibration curve converted to SIE / g or SIE / liter.  

Malta test

A maltase inhibitor unit (MIE) is defined as that Amount of inhibitor that inhibits two maltase units by 50%. A unit of maltase (ME) is the amount of enzyme the one minute under the test conditions given below 1 µmol of maltose splits into 2 µmoles of glucose. The µmoles of glucose formed are generated using the glucose oxidase reaction quantified under conditions where no further maltose cleavage by the maltase more takes place. To carry out the test 0.05 ml of a maltase solution set to 0.060-0.070 ME²) with 1-20 µg inhibitor or 1-20 µl testing solution and mixed with 0.1 sodium maleate buffer pH 6.0 to 0.1 ml of one preheated to 35 ° C 0.05 M maltose solution in 0.1 M sodium maleate buffer pH 6.0 transferred. Incubate 20 minutes at 35 ° C and stop Maltase reaction by adding 1 ml glucose oxidase reagent¹) and incubate at 35 ° C for a further 30 minutes.

Then 1 ml of 50% H₂SO₄ is added and at 545 against measured a corresponding blank value.

1) The glucose oxidase reagent is dissolved by dissolving 2 mg glucose oxidase (grade I) in 100 ml 0.565 m Tris-HCl buffer pH 7.0 and then adding 1 ml detergent solution (2 g Triton X 100 + 8 g 95% ethanol pa) , 1 ml of dianisidine solution (260 mg of o-dianisidine · 2 HCl in 20 ml of H₂O) and 0.5 ml of 0.1% aqueous peroxidase solution (from Boehringer, Lyophilisat, grade II).
2) Solubilized maltase from pig small intestine mucosa according to B. Borgström, A. Dahlquist, Acta Chem. Scand. 12, (1958), page 1997. Diluted to the appropriate ME content with 0.1 M sodium maleate buffer pH 6.0.

The percentage inhibition of the used is used for evaluation Maltase calculated and from the 50% inhibition point with Converted to MIE / g or MIE / liter using a glucose calibration curve.

Using the method described above, a whole series tested by strains of the Bacillaceae family. In doing so particularly evident in strains of the Bacellus subtilis species Glycoside hydrolase inhibiting activities found.

The frequency with which strains according to the specified method were found to be active inhibitors in the tests proved to be over 5%. Particularly effective strains are listed in Table 1.  

Table 1

Bacillus strains with sucrose inhibitor activity

The strains listed are under the specified DSM Numbers at the German Collection of Microorganisms (DSM), Göttingen and can be obtained from there.

To obtain the glycoside hydrolase inhibitors the strains listed above in those described above Cultivated nutrient solutions. It should be noted that practical each strain has a different one for optimal production, qualitatively and quantitatively different nutrient solution needed.

After incubation for 1-10 days at 15-80 ° C, preferably 24-40 ° C or 50-70 ° C for thermophiles, in shake flasks or in fermenters of different sizes, the cells  separated from the culture solution and depending on the occurrence the inhibitors the effective principle from the culture solution and / or enriched from the cells.

The inhibitors are obtained from the culture broths Lyophilization or precipitation with salts or water-soluble organic solvents (such as lower alcohols and ketones) or by adsorption of the active ingredients Ion exchangers.

The inhibitors are extracted from the cells by extraction with organic solvents, such as. B. alcohols, Ketones, ethers, esters and sulfoxides.

For this, the fermentation batch at 3000-20 000 rpm, preferably 6-10,000 rpm, 10-60 minutes, preferably 30 minutes, centrifuged or filtered, preferably with Pressure and with the aid of filter aids, and thus separated into culture broth and cell residue.

Isolation of the inhibitor from the respective culture broth can be done in different ways:

• a) Concentration of the culture broths under reduced pressure (10-50 Torr) at bath temperatures of 20-100 ° C, preferably 40-80 ° C, to about 1 / 5-1 / 50 of the starting volume. The concentrated extract is filtered or centrifuged and the clear filtrate (the clear Supernatant) possibly lyophilized after prior desalination.  
• b) precipitation of the inhibitors from the culture broth (or the culture broths restricted according to a) by addition of water-soluble organic solvents, such as e.g. B. alcohols or ketones, preferably methanol, Ethanol, acetone, up to a content of 60-90%. Because inactive at low concentration of solvent Accompanying substances are precipitated, is suitable this precipitation method is particularly good for fractional Precipitation to separate unwanted Accompanying substances.
• c) Salting out the inhibitors from the extracts (or the after a) concentrated extracts), e.g. B. with ammonium sulfate, Cooking salt, etc. The precipitate that falls collected by centrifugation or filtration and either washed directly with acetone and ether and dried in vacuo or dialyzed after redissolving in water and lyophilized.
• d) adsorption of the inhibitors on ion exchangers. This Process is suitable for the isolation of such Inhibitors due to their chemical nature charges wear. The inhibitor is desorbed by changing the ionic strength or the pH Value of the elution medium.

example

Inoculate a 1 liter Erlenmeyer flask containing 100 ml of the Contains nutrient solution A, with an inoculation loop with cell material from an overgrown oblique tube of the strain DSM 1060 and shake this flask for 5 days at 28 ° C on one Round shaker, so you get a culture broth 35,000 sucrose inhibitor units per liter.

Nutrient solution A (NL A)

3% soy flour defatted, 3% glycerin, pure, 0.3% CaCO₃ p. A., tap water, pH 6.85 Sterilization: 30 minutes at 121 ° C. pH after sterilization: 7.2.

Nutrient solution B (NL B)

2% corn starch, 1% glucose per inf., 0.5% N-Z- Amine A, 21.0% yeast extract, tap water, pH 5.95 with Na₂CO₃ solution (approx. 10%) to pH 7.2, then 0.4% CaCO₃ p. A. admit. Sterilization: 30 Minutes at 121 ° C, pH after sterilization: 7.6.

In addition to the inhibitor, they are often found in the culture broths unwanted accompanying substances. The separation of these accompanying substances can be done in different ways, e.g. B. by heat denaturation of the accompanying substances in inhibitors, which are heat stable, or by dialysis by appropriate Membranes for low molecular weight inhibitors, the undesired accompanying substances being retained by the membrane or by fractional precipitation or by adsorbing the accompanying substances onto ion exchangers (see above).

Inhibitors are obtained from the cells by repeated extraction of the cells with organic Solvents, preferably twice 10-20 minutes Extraction with 3-5 vol. Acetone (based on the cell moisture volume) and then once for 5-10 minutes  Extraction with ether. The acetone and ether extracts are evaporated to dryness in a vacuum and taken up in water lyophilized.

The new substances dissolve well in water. A group the inhibitors are heat stable at neutral pH values, acid stable (pH 2), alkali stable (pH 12) and dialyzable. These inhibitors are trypsin and pepsin not deactivated, they in turn inhibit the above Enzymes not. They are made with the typical protein colorants not dyeable. According to estimates from the Gel filtration is the molecular weight of these inhibitors over 100 but under 2000.

The best inhibitors in these groups stand out an extremely high, also all previously known sucrose Inhibitor-superior inhibitory activity towards saccharase out.

It is extremely surprising and could not have been foreseen that these inhibitors of organisms of the Bacillus species Subtilis are produced as these are microorganisms generally less than secondary producers are suitable and at most essentially peptide-like Form secondary substances.

It is known that in animals and humans after ingestion of carbohydrate-containing foods and beverages (e.g. cereal, potato starch, fruit, fruit juice, beer, Chocolate) Hyperglycaemia that occurs as a result of  rapid breakdown of carbohydrates by glycoside hydrolases (e.g. saliva and pakreas amylases, maltases, saccharases) according to the following scheme

be effected. These hyperglycemias are in diabetics particularly strong and persistent. In obesity Alimentary hyperglycemia is often a special one strong secretion of insulin, which in turn increases Fat build-up and reduced fat loss leads. In connection Such hyperglycemia occurs in healthy people and obese people as a result of insulin secretion often hypoglycemia. It is known that both Hypoglycaemia and gastric chyme promote the production of gastric juice, which in turn the Development of gastritis, or ulcer ventriculi triggers or favors duondeni.

It is also known that carbohydrates, especially sucrose, are broken down by microorganisms and thereby promoting caries formation.  

Malabsorption of carbohydrates, e.g. B. as a result of intestinal Lack of sucrose, causes diarrhea.

Suitable Doses of a glucosidase inhibitor cause an artificial one Malabsorption and are therefore apt to constipation counteract.

The inhibitors according to the invention are therefore suitable as therapeutics for the following indications:
Obesity, hyperlipoproteinemia, atherosclerosis, diabetes, prediabetes, gastritis, constipation, caries.

To broaden the spectrum of activity, it can be recommended Inhibitors of glycoside hydrolases that are complement each other in their effect, to combine it that they are combinations of the invention Inhibitors with each other or combinations of the inventive Inhibitors deals with already known. For example, it may be appropriate to use the invention Saccharase inhibitors with known amylase inhibitors to combine.

In some cases, combinations of the inhibitors according to the invention with known oral antidiabetics ( β- cytotropic sulfonylurea derivatives and / or blood sugar-effective biguanides) and with blood lipid-lowering active ingredients such as, for example, B. clofibrate, nicotinic acid, cholestyramine and others.

The compounds can be used without dilution, e.g. B. as a powder, or in a gelatin shell or in combination with one Carrier in a pharmaceutical composition be applied.  

Pharmaceutical preparations can be larger or larger contain smaller amount of inhibitor, e.g. B. 0.1% to 99.5%, in combination with a pharmaceutically acceptable non-toxic, inert carrier, the Carrier one or more solid, semi-solid or liquid diluents, fillers and / or non- toxic, inert and pharmaceutically acceptable formulation aid may contain. Such pharmaceutical Preparations are preferably in the form of dosage units before, d. H. physically discrete, one certain amount of units containing the inhibitor equal to a fraction or a multiple of the dose, to bring about the desired inhibitory effect correspond. The dosage units can be 1, 2, 3, 4 or more single doses or 1/2, 1/3 or 1/4 of a single dose contain. A single dose preferably contains a sufficient amount of active ingredient to apply according to a predetermined dosage regimen one or the desired inhibitory effect to achieve, being a whole, a half or a third or a quarter of the daily dose usually once, twice, is administered three or four times a day. Other Therapeutic agents can also be taken. Although the dosage and dosage regimen in each Case should be carefully weighed using thorough professional judgment and in compliance with the Age, weight and condition of the patient who The type and severity of the disease, the dosage usually in a range between about 30 to about 3 times 10⁵ AIE / kg and between about 1 to 1 × 10⁴ SIE / kg of body weight per day. In some cases you will  a sufficient therapeutic effect with a lower dose, while in other cases larger dose will be required.

Oral application can be made using solid and liquid Dosage units are carried out, such as. B. Powders, tablets, coated tablets, capsules, granules, suspensions, Solutions and the like.

Powder is made by crushing the substance in one suitable size and mixing with an also crushed pharmaceutical carrier. Although an edible carbohydrate, such as. B. starch, lactose, Sucrose or glucose usually go with this Purposes are used and also used here , it is desirable to be a non-metabolizable one Carbohydrate such as B. to use a cellulose derivative.

Sweeteners, flavor additives, preservatives, dispersants and colorants can also be used will.

The capsules can be prepared by preparing the above Powder mixture and already formed by filling Gelatine casings are made. The powder mixture can one before the filling process with lubricants, such as. B. silica gel, Talc, magnesium stearate, calcium stearate or solid polyethylene glycol. You can do the mixture also with a disintegrator or solubilizer, such as B. agar, calcium carbonate or sodium carbonate,  move to accessibility when taking the capsule to improve the inhibitor.

The tablets are made, for example, by Preparation of a powder mixture, large or fine-grained, and adding a lubricant and disintegrator. Out This mixture is used to form tablets. A powder mix is prepared by mixing the substance contained in appropriately crushed and replenishes a diluent or another carrier as described above. If necessary, add a binder: e.g. B. carboxymethyl cellulose, alginates, gelatin or polyvinyl pyrrolidones, a solution delay, such as. B. paraffin, a resorption accelerator, such as. B. quaternary Salt and / or an adsorbent, such as. B. Brentonite, Kaolin or dicalcium phosphate. The powder mix can be granulated together with a binder such as e.g. B. syrup, starch paste, acacia mucus, or solutions made of cellulose or polymer materials. Then you press the product through a coarse sieve. As an alternative to this you can use a tablet machine to mix the powder let it run and the resulting irregularly shaped ones Crush pieces down to grain size. So that resulting grains not in the tablet-forming nozzles get stuck, you can add a lubricant, such as B. stearic acid, stearate salt, talc or Mineral oil. This lubricated mixture is then pressed in tablet form. The active ingredients can also with free-flowing inert carriers are combined and are brought directly into tablet form with omission the granulate or chopping steps. You can do that  Provide the product with a clear or opaque protective cover, e.g. B. a shellac coating, a coating Sugar or polymer substances and a polished shell made of wax. Dyes can be added to these coatings so between the different dosage units can be distinguished.

The oral forms of preparation, such as. B. Solutions, syrup and elixirs, can be divided into dosage units produce so that a certain amount of preparation contains a certain amount of active ingredient. Syrup can be made like this that the active ingredient in an aqueous solution, which contains suitable flavors becomes; Elixirs are made using non-toxic, alcoholic Receive carriers. One can use suspensions by dispersing the compound in a non-toxic Represent carrier. Solubilizers and emulsifiers, such as B. ethoxylated isostearyl alcohols and polyoxyethylene sorbitol esters, Preservatives, taste-improving Additives such as B. peppermint oil or saccharin u. Like., Can also be added.

Dosage instructions can be given on the capsule will. In addition, the dosage can be secured that the active ingredient is released with a delay, e.g. B. by Compliance with the active ingredient in polymer substances, waxes or the like

In addition to the pharmaceutical compositions mentioned above can also contain these active ingredients Produce food; for example sugar, bread, potato products,  Fruit juice, beer, chocolate and others Confectionery, and canned goods, such as. B. jam, whereby at least a therapeutically effective amount for these products one of the inhibitors according to the invention was given.

The inhibitors according to the invention also have the property on, in animals the ratio of the proportion unwanted fat to the proportion of the desired low-fat Meat (lean meat) in favor of lean meat to influence to a high degree. This is special Importance for the rearing and keeping of agricultural Farm animals, e.g. B. in pig fattening, however also of considerable importance for rearing and keeping of other farm animals and ornamental animals. The usage The inhibitors can continue to be significant Rationalize animal feeding, both in terms of time, quantity and quality. Since she is a The dwell time will cause some delay in digestion which extends nutrients in the digestive tract, whereby an ad libitum- Feeding is made possible. Furthermore, the Use of the inhibitors according to the invention in many Cases a substantial saving of valuable protein feed.

The active ingredients can practically in all areas animal nutrition as a means of reducing fat deposits as well as to save feed protein will.

The effectiveness of the active ingredients is largely independent on the type and sex of the animals. Especially The active ingredients in animal species prove to be valuable, which at all or in certain stages of life tend to store more fat.

As animals in which the inhibitors to reduce the Fat deposits and / or used to save feed protein can be, for example, the following and ornamental animals: warm-blooded animals, such as cattle, pigs, Horses, sheep, goats, cats, dogs, rabbits, fur animals, e.g. B. mink, chinchilla, other ornamental animals, e.g. B. Guinea pigs and hamsters, laboratory and zoo animals, e.g. B. Rats, mice, monkeys, etc., poultry, e.g. B. broilers, chickens, Geese, ducks, turkeys, pigeons, parrots and canaries, and cold-blooded animals, such as fish, e.g. B. carp, and reptiles, e.g. B. snakes.

The amount of active ingredients that the animals achieve the desired effect is administered, because of the favorable properties of the active ingredients largely varied will. It is preferably about 0.5 mg to 2.5 g, especially 10 to 100 mg / kg feed. The duration of administration can last from a few hours or days to amount to several years. The right amount of active ingredient as well the appropriate duration of administration are narrow Relation to the feeding goal. They hang in particular on the type, age, gender, health status and the way the animals are kept easy to determine by any specialist.  

The active compounds according to the invention are after the animals administered the usual methods. The mode of administration depends in particular on the type, behavior and the general condition of the animals. So can the administration once or several times a day, in regular or irregular intervals, orally. For convenience is oral administration in most cases, especially in the rhythm of food and / or Drink intake of the animals is preferable.

The active ingredients can be formulated as pure substances or Form are administered, the formulated form both as a premix, i.e. in a mixture with non-toxic inert carriers of any kind, as well as part a total ration in the form of a supplement or as To understand the mixture component of a sole compound feed is. The application is also included suitable preparations via drinking water.

The active ingredients can optionally be in formulated form also together with other nutrients and active ingredients, e.g. B. Mineral salts, trace elements, vitamins, protein substances, Energy sources (e.g. starch, sugar, fats), dyes and / or flavorings or other feed additives, e.g. B. growth promoters, administered in a suitable form will. The active ingredients can be be given during or after food.

Oral administration with is recommended the feed and / or drinking water, whereby the Active ingredients of the total amount or only parts of the feed and / or drinking water can be added.  

The active ingredients can be prepared using simple methods Mix as pure substances, preferably in finely divided Form or in formulated form in a mixture with edible, non-toxic carriers, if necessary also in the form of a premix or a feed concentrate, the Food and / or drinking water can be added.

The feed and / or drinking water can, for example Active substances according to the invention in a concentration of about 0.001 to 5.0%, in particular 0.02 to 2.0% (weight) contain. The optimal level of concentration of the Active ingredient in the feed and / or drinking water is in particular depending on the amount of feed and / or drinking water intake of animals and can be easily done by any professional be determined.

The type of feed and its composition is here irrelevant. It can all common, commercially available or special feed compositions used be, which is preferably the usual, for a balanced Nutrition necessary balance of energy and protein substances, including vitamins and minerals, contain. For example, the feed can be composed from vegetable substances, e.g. B. oil cake pods, Grain meal, grain by-products, but also from Hay, fermentation feed, beets and other fodder plants animal substances, e.g. B. meat and fish products, Bone meal, fats, vitamins, e.g. B. A-, D-, E-, K- and B- Complex, as well as special protein sources, e.g. B. yeasts, as well certain amino acids and minerals and trace elements, such as B. phosphorus and iron, zinc, manganese, Copper, cobalt, iodine etc.  

Premixes can preferably be about 0.1 to 50%, in particular 0.5 to 5.0% (weight) of the active ingredients of the formula I. in addition to any edible carriers and / or mineral salts, e.g. B. carbonated lime, contain and will manufactured according to the usual mixing methods.

Compound feeds preferably contain 0.001 to 5.0%, in particular 0.02 to 2.0% (weight) of the active ingredients of the formula I. in addition to the usual raw material components of a compound feed, e.g. B. cereal meal or by-products, oil cake meal, animal protein, minerals, trace elements and vitamins. You can use the usual mixing methods getting produced.

Preferably in premixes and compound feed Active ingredients, if necessary, also by their surface covering suitable means, e.g. B. with non-toxic Waxing or gelatin, from air, light and / or moisture to be protected.

Example of the composition of a finished compound feed, for poultry, which contains an active ingredient according to the invention:
200 g wheat, 340 g maize, 360.3 g soybean meal, 60 g beef tallow, 15 g dicalcium phosphate, 10 g calcium carbonate, 4 g iodized table salt, 7.5 g vitamin-mineral mixture and 3.2 g active ingredient premix carefully mixing 1 kg of feed.

The vitamin-mineral mixture consists of:
6000 IU vitamin A, 1000 IU vitamin D₃, 10 mg vitamin E, 1 mg vitamin K₃, 3 mg riboflavin, 2 mg pyridoxine, 20 mcg vitamin B₁₂, 5 mg calcium pantothenate, 30 mg nicotinic acid, 200 mg choline chloride, 200 mg MnSO₄ × H₂O , 140 mg ZnSO₄ × 7 H₂O, 100 mg FeSO₄ × 7 H₂O and 20 mg CuSO₄ × 5 H₂O. The active ingredient premix contains the inhibitor according to the invention in the desired amount, e.g. B. 1600 mg, and additionally 1 g of DL-methionine and so much soybean meal that 3.2 g of premix are formed.

Example of the composition of a pig feed containing an active ingredient of formula I:
630 g of feed grain meal (composed of 200 g maize meal, 150 g barley meal, 150 g oat meal and 130 g wheat meal), 80 g fish meal, 60 g soy meal, 58.8 g tapioca meal, 38 g brewer's yeast, 50 g vitamin mineral Mix for pigs (composition, e.g. for chick feed), 30 g linseed cake flour, 30 g corn gluten feed, 10 g soybean oil, 10 g sugarcane molasses and 2 g active ingredient premix (composition e.g. for chick feed) after thorough mixing 1 kg of feed.

The specified feed mixtures are preferably for rearing and fattened by chicks or pigs, they can, however, in the same or a similar composition can also be used for rearing and fattening other animals.  

As already mentioned, the inhibitors can be used individually or but also used in any mixtures with one another are, both the pure active ingredients and the crude active ingredients obtained in the preparation, if appropriate after a rough cleaning.

Claims (8)

1. Inhibitors of glycoside hydrolases from the strains DSM 1060, DSM 1061, DSM 1062, DSM 1063, DSM 1064, DSM 1065, DSM 1066 and DSM 1067, except l-deoxynojirimycin. 2. Inhibitors of glucosidases in the digestive tract take effect from the strains DSM 1060, DSM 1061, DSM 1062, DSM 1063, DSM 1064, DSM 1065, DSM 1066, DSM 1067, except l-deoxynojirimycin. 3. Inhibitors for a -glucose doses of the digestive tract from the strains DSM 1060, DSM 1061, DSM 1062, DSM 1063, DSM 1064, DSM 1065, DSM 1066 and DSM 1067, with the exception of l-deoxynojirimycin. 4. Saccharase inhibitors from the strains DSM 1060, DSM 1061, DSM 1062, DSM 1063, DSM 1064, DSM 1065, DSM 1066 and DSM 1067, except l-deoxynojirimycin. 5. Process for the preparation of the inhibitors according to Claims 1 to 4, characterized in that one the strains DSM 1060, DSM 1061, DSM 1062, DSM 1063, DSM 1064, DSM 1065, DSM 1066 and DSM 1067 in usual Nutrient solutions at temperatures from about 15 to about 80 ° C for about 1 to about 8 days with aeration in usual Fermentation vessels cultivated the cells thrown off and the inhibitors from the culture broth  or the cell extracts through normal cleaning operations isolated. 6. Use of the inhibitors according to claims 1 to 4 to influence the carbohydrate metabolism of Humans and animals. 7. Use of the inhibitors according to claims 1 to 4 as therapeutic agents for the treatment of obesity and / or atherosclerosis and / or diabetes and / or Prediabetes and / or gastritis and / or constipation and / or tooth decay. 8. Use of the inhibitors according to claims 1 to 4 in animals to avoid unwanted fat deposits and to achieve an increased meat intake as well as for better feed utilization.